1. Field of the Invention
The present invention relates to generally to gene promoters. More specifically, the present invention relates to the thymosin xcex215 promoter and use of this promoter in diagnostic and therapeutic applications as well as in assays for compounds that modulate thymosin xcex215 expression.
2. Background
Beta thymosins are a family of closely related, highly polar 5 kDa polypeptides. All vertebrates studied and some invertebrates are known to contain one or often two beta thymosins (Nachmias VT, Curr. Opin. Cell Biol., 1993;56-62). Thymosin xcex215 was recently uncovered in a search for genes with increased expression in motile as compared to poorly motile Dunning rat prostatic carcinoma cell lines (Bao, et al., Nature Medicine, 1996; 1322-1328). The protein, which is 5300 Da, was designated thymosin xcex215 because of its approximately 60% homology with other members of the beta thymosin family.
Thymosin xcex24 is the most abundant beta thymosin in most mammalian tissue and is the best-studied member of this family. Current understanding is that thymosin xcex24 sequesters a large pool of monomeric actin that is accessible to be released as needed for polymerization of actin filaments (Cassimeris, et al., J. Cell Biol., 1992;119:1261-70, Weber, et al., Biochemistry, 1992;31:6179-85, Nachmias, et al., Eur. J. Cell Biol. 1993;61:314-20). Microinjection or overexpression of thymosin xcex24 has been shown to cause disassembly of actin stress fibers (Sanders, et al., Proc. Natl. Acad. Scl. USA, 1992;89:4678-82; Sanger, et al., Cell Motil. Cytoskeleton, 1995;31:307-22; Yu, et al., Cell Motil Cytoskeleton, 1994;27: 13-25). Like thymosin xcex24, thymosin xcex215 binds monomeric actin and inhibits actin polymerization confirming its place in the beta thymosin family. Thymosin xcex215 also appears to positively regulate cell motility as transfection of antisense thymosin xcex215 into motile rat prostatic carcinoma lines impairs cell motility in a Boyden chamber apparatus (Bao, et al.).
The putative role of beta thymosin in modulation of the actin cytoskeleton through monomer sequestration suggests that they may be involved in cell differentiation, carcinogenesis and metastasis. In some cell lines, increased thymosin xcex24 protein or mRNA has been shown to correlate with differentiation, while in most others it has not (Safer, et al., Bioessays, 1994; 16:473-9). In human tumors, thymosin xcex24 mRNA has been shown to be increased in hairy cell leukemia and reduced in some lymphomas (Otero, et al., Biochem Biophys. Acta., 1993; 1176:59-63). Two out of three metastatic colorectal carcinomas showed decreased thymosin xcex24 mRNA compared to non-metastatic tumors with the third metastatic tumor showing little change (Yamamoto, et al., Biochem Biophys. Res. Commun, 1993; 193:706-10). Thymosin xcex210 mRNA levels are increased in renal cell carcinomas (Hall, AK, Ren. Fail, 1994; 16:243-54, Hall, AK, Cell Mo. Biol. Res. Commun, 1995;41:167-80), and thymosin xcex210 up-regulation was shown to correlate with the metastatic potential of melanomas (Weterman, et al., Int. J Cancer, 1993; 53:278-84). The expression of each thymosin beta family member is independently regulated. Consequently different family members may be independently elevated or decreased in particular tumor types. As thymosin xcex215 has only recently been described, it is less well characterized. It is present in very few normal adult tissues but was shown to be up-regulated in metastatic human prostate cancers at both the mRNA and protein level as compared to less metastatic prostate cancers (Bao, et al.). Immunostaining of human prostate cancer cases revealed a general correlation between Gleason grade and thymosin xcex215 expression, with high grade tumors (Gleason grade 8-10) showing increased staining compared to low grade tumors (Gleason grade 2-5).
Thymosin xcex215 was also up-regulated in 15/20 human pancreatic tumors. Additionally, thymosin xcex215 has been shown to be up-regulated in malignant as compared with benign breast tissue and may represent an early marker for breast malignancy (Gold, et al., Modem Pathology 1997, In press). The restricted distribution of thymosin xcex215 may allow targeted destruction of malignant tumors expressing the protein e.g., prostate, breast and pancreatic tumors.
The present invention provides a thymosin xcex215 promoter. The promoter comprises the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof capable of expressing an operably linked DNA. A DNA sequence having nucleotides xe2x88x92400 to +1 of FIG. 1 (nucleotide 153-572 of SEQ ID NO: 1) is a preferred fragment.
The invention further provides a vector having the promoter of the present invention operably linked to a DNA encoding a gene product and host cell containing such vectors. These gene products can be marker genes, toxins, suicide genes, viral genes, ribozymes, intrabodies, antisense, or another heterologous gene product.
The invention also provides novel assays for identifying compounds useful in the treatment of malignancies involving modulation of thymosin xcex215 expression, e.g., breast, pancreas and prostate cancer. Preferred compounds identified through assays of the invention can modulate, particularly inhibit, thymosin xcex215. A preferred assay comprises transfecting a host cell with a vector containing the thymosin xcex215 promoter operably linked to a gene encoding a reporter or marker protein; contacting the cell with a test compound and measuring reporter protein expression.
The invention also provides a method of human gene therapy for treating malignancies involving up-regulation of thymosin xcex215 expression, e.g., breast, pancreas and prostate cancer. The method comprises administering to a human in need thereof an expression vector comprising the thymosin xcex215 promoter operably linked to a DNA encoding a gene product the expression of which by the cell expressing thymosin xcex215 , i.e., the cancer cells, inhibits the growth of the cell or results in the cells death. Such gene products include, for example, toxins, suicide genes, ribozymes, intrabodies, or antisense DNA or RNA.
Other aspects of the invention are disclosed infra.